The present invention is to the discovery of a new method to block excessive or inappropriate apoptosis in a mammal.
It has been recognized for over a century that there are different forms of cell death. One form of cell death, necrosis, is usually the result of severe trauma and is a process that involves loss of membrane integrity and uncontrolled release of cellular contents, often giving rise to inflammatory responses. In contrast, apoptosis is a more physiological process that occurs in a controlled manner and is generally non-inflammatory in nature. For this reason apoptosis is often referred to as programmed cell death. The name itself (apoptosis: Greek for xe2x80x9cdropping offxe2x80x9d, for example leaves from trees) implies a cell death that is part of a normal physiological process (Kerr et al., Br. J. Cancer, 26: 239-257 (1972)).
Apoptosis appears to be a carefully controlled series of cellular events which ultimately leads to death of the cell. This process for elimination of unwanted cells is active and requires expenditure of cellular energy. The morphological characteristics of apoptosis include cell shrinkage and loss of cell-cell contact, condensation of nuclear chromatin followed by fragmentation, the appearance of membrane ruffling, membrane blebbing and apoptotic bodies. At the end of the process, neighboring cells and macrophages phagocytose the fragments from the apoptotic cell. The process can be very fast, occurring in as little as a few hours (Bright et al., Biosci. Rep., 14: 67-82 (1994)).
The best defined biochemical event of apoptosis involves the orderly destruction of nuclear DNA. Signals for apoptosis promote the activation of specific calcium- and magnesium-dependent endonucleoases that cleave the double stranded DNA at linker regions between nucleosomes. This results in production of DNA fragments that are multiples of 180-200 base pair fragments (Bergamaschi et al., Haematologica, 79: 86-93 (1994); Stewart, JNCI, 86: 1286-1296 (1994)). When examined by agarose gel electrophoresis, these multiple fragments form a ladder pattern that is characteristic for most cells undergoing apoptosis.
There are numerous stimuli that can signal cells to initiate or promote cellular apoptosis, and these can be different in different cells. These stimuli can include glucocorticoids, TNFa, growth factor deprivation, some viral proteins, radiation and anticancer drugs. Some of these stimuli can induce their signals through a variety of cell surface receptors, such as the TNF/nerve growth factor family of receptors, which include CD40 and Fas/Apo-1 (Bright et al., supra). Given this diversity in stimuli that cause apoptosis it has been difficult to map out the signal transduction pathways and molecular factors involved in apoptosis. However, there is evidence for specific molecules being involved in apoptosis.
The best evidence for specific molecules that are essential for apoptosis comes from the study of the nematode C. elegans. In this system, genes that appear to be required for induction of apoptosis are Ced-3 and Ced-4. These genes must function in the dying cells and, if either gene is inactivated by mutation, cell death fails to occur (Yuan et al., Devel. Biol., 138: 33-41 (1990)). In mammals, genes that have been linked with induction of apoptosis include the proto-oncogene c-myc and the tumor suppresser gene p53 (Bright et al., supra; Symonds et al., Cell, 78: 703-711 (1994)).
In this critical determination of whether or not to undergo apoptosis, it is not surprising that these are genes that program for proteins that inhibit apoptosis. An example in C. elegans is Ced-9. When it is abnormally activated, cells survive that would normally die and, conversely, when Ced-9 is inactivated cells die that would normally live (Stewart, B. W., supra). A mammalian counterpart is bcl-2, which had been identified as a cancer-causing oncogene. This gene inhibits apoptosis when its product is overexpressed in a variety of mammalian cells, rendering them less sensitive to radiation, cytotoxic drugs and apoptotic signals such as c-myc (Bright et al., supra). Some virus protein have taken advantage of this ability of specific proteins to block apoptosis by producing homologous viral proteins with analogous functions. An example of such a situation is a protein produced by the Epstein Barr virus that is similar to bcl-2, which prevents cell death and thus enhances viral production (Wells et al., J. Reprod. Fertil., 101: 385-391 (1994)). In contrast, some proteins may bind to and inhibit the function of bcl-2 protein, an example being the protein bax (Stewart, B. W., supra). The overall picture that has developed is that entry into apoptosis is regulated by a careful balancing act between specific gene products that promote or inhibit apoptosis (Barinaga, Science, 263: 754-756 (1994).
Apoptosis is an important part of normal physiology. The two most often sited examples of this are fetal development and immune cell development. In development of the fetal nervous system, over half of the neurons that exist in the early fetus are lost by apoptosis during development to form the mature brain (Bergamaschi et al., Haematologica, 79: 86-93 (1994)). In the production of immune competent T cells (and to a lesser extent evidence exists for B cells), a selection process occurs that eliminates cells that recognize and react against self. This selection process is thought to occur in an apoptotic manner within areas of immune cell maturation (Williams, G. T., J. Pathol., 173: 1-4 (1994); Krammer et al., Curr. Opin. Immunol., 6: 279-289 (1994)).
Dysregulation of apoptosis can play an important role in disease states, and diseases can be caused by both excessive or too little apoptosis occurring. An example of diseases associated with too little apoptosis would be certain cancers. There is a follicular B-cell lymphoma associated with an aberrant expression of functional bcl-2 and an inhibition of apoptosis in that cell (Bergamaschi et al., supra). There are numerous reports that associate deletion or mutation of p53 with the inhibition of apoptosis and the production of cancerous cells (Kerr et al., Cancer, 73: 2013-2026 (1994); Ashwell et al., Immunol. Today, 15: 147-151, (1994)). In contrast, one example of excessive or inappropriate apoptosis is the loss of neuronal cells that occurs in Alzheimer disease, possible induced by bamyloid peptides (Barr et al., BioTechnology, 12: 487-493 (1994)). Other examples include excessive apoptosis of CD4+ T cells that occurs in HIV infection, of cardiac myocytes during infarction/reperfusion and of neuronal cells during ischemia (Bergamaschi et al., supra); Barr et al., supra).
Some pharmacological agents attempt to counteract the lack of apoptosis that is observed in cancers. Examples include topoisomerase II inhibitors, such as the epipodophyllotoxins, and antimetabolites, such as ara-c, which have been reported to enhance apoptosis in cancer cells (Ashwell et al., supra). In many cases with these anti-cancer drugs, the exact mechanism for the induction of apoptosis remains to be elucidated.
In the last few years, evidence has built that ICE and proteins homologous to ICE (Caspases) play a key role in apoptosis. This area of research has been spurred by the observation of homology between the protein coded by Ced-3, a gene known to be critical for C. Elegans apoptosis, and ICE (Caspase 1). These two proteins share 29% amino acid identity, and complete identity in the 5 amino acid portion thought to be responsible for protease activity (QACRG) (Yuan et al., Cell, 75: 641-652 (1993)). Additional homologies are observed between ICE and the product of the nedd-2 gene in mice, a gene suspected of involvement in apoptosis in the developing brain (Kumar et al., Genes Dev., 8: 1613-1626 (1994)) and Ich-1 (Caspase 2) and CPP32 (Caspase 3), human counterparts of nedd-2 isolated from human brain cDNA libraries (Wang et al., Cell, 78: 739-750 (1994); Fernandes-Alnemiri et al., J. Biol. Chem., 269: 30761-30764 (1994)).
Further proof for the role of these proteins in apoptosis comes from transfection studies. Over expression of murine ICE caused fibroblasts to undergo programmed cell death in a transient transfection assay (Miura et al., Cell, 75: 653-660 (1993)). Cell death could be prevented by point mutations in the transfected gene in the region of greatest homology between ICE and Ced-3. As very strong support for the role of ICE in apoptosis, the authors showed that ICE transfection-induced apoptosis could be antagonized by overexpression of bcl-2, the mammalian oncogene that can prevent programmed cell death (Miura et al., supra). Additional experiments were performed using the crmA gene. This gene of the cowpox virus encodes a serpin protein, a family of proteins that are inhibitors of proteases (Ray et al., Cell, 69: 597-604 (1992)). Specifically, the protein of crmA has been shown to inhibit processing of pro-interleukin-1b by ICE. (Gagliardini et al. Science, 263: 826-828 (1994)) showed that microinjection of the crmA gene into dorsal root ganglion neurons prevented cell death induced by nerve growth factor deprivation. This result shows that ICE is involved in neuronal cell apoptosis. A more direct demonstration of ICE involvement comes from experiments in which ICE transfection is coupled with the co-expression of crmA, demonstrating a crmA-induced suppression of the ICE-induced apoptosis response (Miura et al., supra; Wang et al., supra).
In addition to ICE, researchers have examined the ability of Caspases to promote apoptosis. (Kumar et al. supra) demonstrated that over expression of nedd-2 in fibroblasts and neuroblastoma cells resulted in cell death by apoptosis and that this apoptosis could also be suppressed by expression of the bcl-2 gene. Most recently, Wang et al., (Wang et al., supra) examined the over expression of Ich-1 in a number of mammalian cells. Expression resulted in cell apoptosis, which could be antagonized by bcl-2 coexpression. Mutation of a cysteine residue, contained within the QACRG motif and presumed to be critical for protease function, to serine abolished apoptotic activity.
Further evidence for a role of a cysteine protease in apoptosis comes from a recent report by Lazebnik et al. (Nature, 371: 346-347 (1994)). These authors have used a cell-free system to mimic and study apoptosis. In their system there is a protease activity that cleaves the enzyme poly(ADP-ribose) polymerase at a site identical to a cleavage site in pre-interleukin-1b. However, this yet to be isolated protease and ICE appear to be different and to act on different substrate proteins. Blockade of protease activity in the system, using non-selective cysteine protease inhibitors, resulted in inhibition of apoptosis.
Taken together, the above evidence provides striking involvement of Caspases in the induction of apoptosis in mammalian cells. Brain interleukin-1 has been reported to be elevated in Alzheimer disease and Down syndrome (Griffin et al., Proc. Natl. Acad. Sci. U.S.A., 86: 7611-7615 (1989)). There are also reports that interleukin-1 can increase the mRNA and production of b-amyloid protein, a major component of senile plaques in Alzheimer disease as well as in brains of people with Down syndrome and with aging (Forloni et al., Mol. Brain Res., 16: 128-134 (1992); Buxbaum et al., Proc. Natl. Acad. Sci. U.S.A., 89: 10075-10078 (1992); Goldgaber et al., Proc. Natl. Acad. Sci. U.S.A., 86: 7606-7610 (1989)). These reports can be viewed as additional evidence for the involvement of ICE in these diseases and the need for use of a novel therapeutic agent and therapy thereby.
To date, no useful therapeutic strategies have blocked excessive or inappropriate apoptosis. In one patent application, EPO 0 533 226 a novel peptide structure is disclosed which is said to be useful for determining the activity of ICE, and therefore useful in the diagnoses and monitoring of IL-1 mediated diseases. Therefore, a need exists to find better therapeutic agents which have non-toxic pharmacological and toxicological profiles for use in mammals. These compounds should block excessive or inappropriate apoptosis cells, and hence provide treatment for diseases and conditions in which this condition appears.
The present invention is to the novel compounds of Formula (I), their pharmaceutical compositions, and to the novel inhibition of Caspases for use in the treatment of apoptosis, and disease states caused by excessive or inappropriate cell death. The compounds of Formula I are most effective in inhibiting Caspases three and seven.
Another aspect of the present invention is to a pharmaceutical composition comprising a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or diluent.
Another aspect of the present invention is to a method for the treatment of diseases or disorders associated with excessive IL-1b convertase activity, in a mammal in need thereof, which method comprises administering to said mammal an effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.
Another aspect of the present invention is to a method of preventing or reducing apoptosis in a mammal, preferably a human, in need of such treatment which method comprises administering to said mammal or human an effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.
Another aspect of the present invention is to a method of blocking or decreasing the production of IL-1b and/or TNF, in a mammal, preferably a human, in need of such treatment which method comprises administering to said mammal or human an effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.
The compounds of Formula I are represented by the structure 
wherein
R1 and R2 together with the nitrogen to which they are attached form a 4 to 7 membered ring;
R3 and R4 together with the nitrogen to which they are attached form a 4 to 7 membered ring;
R5 is 
xe2x80x83preferably methyl.
Preferably R1 and R2 and R3 and R4 are joined to form a five membered nitrogen containing ring.
Compounds exemplified by Formula (I) include, but are not limited to:
(S,S)-4-[1-(2-Methoxymethyl)pyrrolidinyl)sulfonyl]-2-[1-(2-methoxymethyl)pyrrolidino-ethanedione]-aniline
(S,S)-4-[1-(2-Phenoxymethyl)pyrrolidinyl)sulfonyl]-2-[1-(2-methoxymethyl)pyrrolidino-ethanedione]-aniline
(S,S)-4-[1-(2-(Phenoxymethyl)pyrrolidinyl)sulfonyl]-2-[1-(2-methoxymethyl)pyrrolidino-ethanedione]-N-benzoylaniline
(S,S)-4-[1-(2-(Phenoxymethyl)pyrrolidinyl)sulfonyl]-2-[1-(2-methoxymethyl)pyrrolidino-1,2-ethanedione]-N-methylaniline
The term xe2x80x9cexcessive IL-1b convertase activityxe2x80x9d is used herein to mean an excessive expression of the protein, or activation of the enzyme.
The term xe2x80x9cC1-6 alkylxe2x80x9d or xe2x80x9calkylxe2x80x9d is used herein to mean both straight and branched chain radicals of 1 to 6 carbon atoms, unless the chain length is otherwise specified, including, but not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, and the like.
The present invention contains the inhibition of Caspases by compounds of Formula (I). What is meant by the term xe2x80x9cCaspasesxe2x80x9d are fragment, homologs, analogs and derivatives of the polypeptides Interleukin-1b converting enzyme (or convertase). These analogs are structurally related to the Caspase family. They generally encode a protein (s) which exhibits high homology to the human ICE over the entire sequence. Preferably, the pentapeptide QACRG is conserved. The Caspases, which may include many natural allelic variants (such as substitutions, deletion or addition of nucleotides) does not substantially alter the function of the encoded polypeptide. That is they retain essentially the same biological function or activity as the ICE protease, although it is recognized that the biological function may be enhanced or reduced activity. The suitable activity is not IL-1b convertase activity, but the ability to induce apoptosis or involved in programmed cell death in some manner. Suitable Caspases encompasses within this invention are those described in PCT US94/07127 filed Jun. 23, 1994, and in U.S. Ser. No. 08/334,251, filed Nov. 1, 1994, whose disclosures are incorporated herein by reference in their entirety.
The term xe2x80x9cblocking or inhibiting, or decreasing the production of IL-1b and/or TNFxe2x80x9d as used herein refers to:
a) a decrease of excessive levels, or a down regulation, of the cytokine in a human to normal or sub-normal levels by inhibition of the in vivo release of the cytokine; or
b) a down regulation, at the genomic level, of excessive in vivo levels of the cytokine (IL-1 or TNF) in a human to normal or subnormal levels; or
c) a down regulation, by inhibition of the direct synthesis of the cytokine (IL-1, or TNF) as a postranslational event, or
d) a down regulation, at the translational level, of excessive in vivo levels of the cytokine (IL-1, or TNF) in a human to normal or sub-normal levels.
Compound of the present invention may be synthesized in accordance with the schemes illustrated below. 
5-Isatinsulfonic acid, sodium salt (1) is treated with phosphorus oxychloride in organic solvents such as sulfolane at temperatures ranging from 50-80xc2x0 C. in order to obtain 5-chlorosulfonylisatin (2) (Martinez, F; Naarmnann; H, Synth. Met., 1990, 39, 195), a direct precursor to the novel compounds of this invention. Treatment of the chlorosulfonyl derivative 2 with an excess of a secondary amine in organic solvents such as tetrahydrofuran, methylene chloride or dimethylformamide yields the 4-alkylaminosulfonyl-2-alkylglyoxylamidoaniline 3. Alternatively, treatment of the chlorosulfonyl derivative 2 with a slight excess ( less than 1.3 eq) of a secondary amine in organic solvents such as tetrahydrofuran, methylene chloride or dimethylformamide with a tertiary amine base such as triethylamine yields the 5-alkylaminosulfonylisatin 4. The 5-alkylaminosulfonylisatin derivative 4 is treated with a secondary amine base in organic solvents such as tetrahydrofuran, methylene chloride, dimethylformamide, or methanol in temperatures ranging from 25-60 xc2x0 C. to yield the 4-alkylaminosulfonyl-2-alkylglyoxylamidoaniline 5. The 4-alkylaminosulfonyl-2-alkylglyoxylamidoaniline 5 is acylated with an acyl chloride in pyridine to yield the 4-alkylaminosulfonyl-2-alkylglyoxylamido-N-acylanilide 6. Additionally, the 5-alkylaminosulfonylisatin 4 is alkylated in the presence of a base such as K2CO3 or NaH with a halide such as methyl iodide or benzyl bromide in an organic solvent to give 1-alkyl-5alkylaminosulfonlyisatin 7. The 1-alkyl-5alkylaminosulfonlyisatin 7 is treated with a secondary amine base in organic solvents such as tetrahydrofuran, methylene chloride, dimethylformamide, or methanol to yield the 4-alkylaminosulfonyl-2-alkylglyoxylamido-N-alkyl-aniline 8.